Does non-digested RNA in cDNA sample have an effect on RT-PCR quantification? - (Feb/16/2007 )

Hello everyone!

I was thinking about something. Suppose that you reverse transcribed your total RNA with an RNase H(-) RT to obtain cDNAs for longer transcripts. Then, if you directly use this cDNA for an RT-PCR reaction, you put into the tube both the cDNA and the total RNA.

What I want to know is, is it possible for PCR primers to anneal to transcripts at 58 C? If then, what I amplify is both the RNA and the cDNA, right?

In that case, should I digest the RNA after the RT reaction?

-jahan-

hi,

normally when you use kits to reverse transcribe RNA there is an RNAse digestion step in the end. The primer can also bind RNA.Cheers

-Bomber-

Amplification of some PCR targets (>1 kb) may require the removal of RNA complementary to the cDNA.